US8729244B2 - Method for purifying factor B - Google Patents

Method for purifying factor B Download PDF

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US8729244B2
US8729244B2 US13/510,140 US201013510140A US8729244B2 US 8729244 B2 US8729244 B2 US 8729244B2 US 201013510140 A US201013510140 A US 201013510140A US 8729244 B2 US8729244 B2 US 8729244B2
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factor
fraction
sepharose
exchange chromatography
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US20120232253A1 (en
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Michel Poulle
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LFB SA
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/644—Coagulation factor IXa (3.4.21.22)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21022—Coagulation factor IXa (3.4.21.22)

Definitions

  • the invention relates to a method for purifying factor B from human blood plasma.
  • Complement plays an essential role in the organism's defence against infectious agents and in the inflammatory process. It represents a helper system for immunity, in particular for the humoral response and for the innate response.
  • Complement comprises a set of approximately 30 plasma and sometimes membrane proteins, synthesized essentially by the liver and the macrophages and which can be activated by proteolytic cascades. It comprises both plasma proteins, numerous different surface cell receptors, some present on inflammatory cells and others on immune system cells, and also regulatory membrane proteins which protect the host cells against self-attack.
  • the complement plasma proteins operate either as enzymes, or as binding proteins, or as regulators (inhibitors or activators).
  • Complement factor B is a component of the alternative complement activation pathway. It circulates in the blood as a protein composed of a single polypeptide chain with a molecular weight of 93 kDa. During activation of the alternative pathway, factor B is cleaved by complement factor D, resulting in an inactive Ba chain (30 kDa) and in the Bb catalytic subunit (63 kDa).
  • the Bb active subunit is a serine protease which associates with C3b to form the C3 convertase of the alternative pathway (C3bBb).
  • the Bb subunit is involved in the proliferation of pre-activated B lymphocytes, while the Ba chain inhibits their proliferation.
  • Factor B preparations would be of use in the treatment of factor B deficiencies, but for the moment, no preparation of this type is commercially available.
  • the invention provides a method for purifying factor B, comprising the steps consisting in:
  • the method may also comprise at least one viral inactivation treatment, preferably via the action of a solvent and detergent, and/or at least one viral removal treatment, preferably by nanofiltration.
  • a subject of the invention is also a method for obtaining a factor B preparation for therapeutic use, which comprises:
  • a subject of the invention is also the factor B preparation that can be obtained by means of this method.
  • said preparation is in lyophilized form.
  • the method of the invention has many advantages: it is industrializable, and makes it possible to purify factor B from various production intermediates, and with a high degree of purity.
  • FIG. 1 shows a scheme representing the purification method steps followed in Example 1.
  • factor B is intended to mean any protein having the amino acid sequence of native human factor B.
  • factor B also comprises the natural allelic variations and/or the isoforms of factor B that are found naturally, and any form or degree of glycosylation or other post-translational modification.
  • factor B homologues or derivatives which have the same or a greater biological activity compared with the activity of the wild-type form and/or which have a sequence identity of at least 80%, preferably at least 85%, more preferably at least 90%.
  • biological activity of factor B includes the ability to activate C3 convertase.
  • the activity of factor B can be measured in various ways, well known to those skilled in the art.
  • a chromatography consists in bringing factor B in solution into contact
  • the supernatant of a cryoprecipitate of blood plasma optionally mixed with a plasma fraction not retained on an anion exchange chromatography of DEAE-Sephadex type is used as starting material. It is also possible to use the supernatant resulting from an ethanol precipitation. A plasma fraction not retained on an anion exchange chromatography can also be used.
  • Heparin-like affinity chromatography uses a matrix or a support, which is most commonly agarose gel, onto which heparin or heparin derivatives or mimetics are grafted.
  • heparin-like ligands mention may in particular be made of the following ligands: chondroitin sulphate, heparan sulphate, dermatan sulphate, keratan sulphate, or synthetic oligomers, for example cellubiose sulphate ester (MATREXTM Cellufine Sulfate), or therapeutic analogues of heparin, for example sulphated oligosaccharides.
  • the pH is adjusted to a value of 6, for example by equilibrating the column with a 20 mM sodium phosphate buffer solution, with an osmolality of 60 ⁇ 5 mOsm/kg, and a pH 6.0 ⁇ 0.1.
  • Example 1 An example of a particular protocol is presented in Example 1.
  • This step makes it possible to bind factor B and antithrombin III (AT III), and to elute them separately.
  • Cation exchange chromatography uses a matrix, most commonly of agarose or polymeric type, onto which are grafted ligands such as carboxymethyl (CM), sulpho-propyl (SP) or methyl sulphonate (S).
  • Cation exchange supports are commercially available: mention may, for example, be made of CM-Sepharose, SP-Sepharose and S-Sepharose (GE Healthcare), Toyopearl CM-650 and Toyopearl SP-650 (Tosoh Biosciences), Fractogel EMD SO3 and Fractogel EMD COO (Merck KGaA), Macro-Prep CM Support and Macro-Prep High S Support (BioRad), and CM HyperD, S HyperD, CM Trisacryl, SP Trisacryl and SP Sperodex (all Pall) supports.
  • SP Sepharose FF is used.
  • An example of a particular protocol is presented in Example 1.
  • Factor B can, under certain conditions, bind to anion exchange resins, and be desorbed therefrom using buffer systems with a high ionic strength.
  • Anion exchange chromatography uses a matrix, most commonly of agarose or polymeric type, onto which are grafted ligands such as diethylaminoethyl (DEAE), quaternary aminoethyl (QAE) or quaternary ammonium (Q).
  • DEAE diethylaminoethyl
  • Q quaternary aminoethyl
  • Q quaternary ammonium
  • Anion exchange supports are commercially available: mention may, for example, be made of DEAE-Sepharose and Q-Sepharose (GE Healthcare), Toyopearl DEAE-650 and Toyopearl Super Q-650 (Tosoh Biosciences), Fractogel EMD DEAE and Fractogel EMD TMAE (Merck KGaA), Macro-Prep DEAE Support and Macro-Prep High Q Support (BioRad), and DEAE HyperD, Q HyperD, DEAE Trisacryl and DEAE Sperodex (all Pall) supports.
  • Q Sepharose FF is preferably used.
  • Example 1 An example of a particular protocol is presented in Example 1.
  • the factor B is eluted by increasing the ionic strength of the anion exchange chromatography equilibration buffer.
  • the pH of the blood plasma fraction containing factor B and obtained in step (i) is adjusted so as to be included in the range of from pH 5.5 to pH 6.5 and preferably so as to be equal to pH 6.0.
  • the pH of the eluted fraction from step (ii) which is then diluted before step (iii) is adjusted so as to be included in the range of from pH 5.5 to pH 7.5.
  • the pH of the eluted fraction from step (iii) which is then diluted before step (iv) is adjusted so as to be included in the range of from 5.5 to 7.5.
  • the factor B purification comprises the steps consisting in:
  • the method is generally continued by means of steps of formulation, concentration, and then filtration of the factor B concentrate.
  • the method for producing a factor B preparation according to the invention can comprise the following steps:
  • the chromatography of step (ii) is the only heparin-like affinity chromatography, the method not comprising any additional heparin-like affinity chromatography step.
  • the method of the invention contains no chromatography other than those provided for in steps (ii) to (iv) defined above.
  • the method of the invention can optionally comprise additional steps, for example other anion or cation exchange chromatographies, but also, where appropriate, one or more hydrophobic interaction chromatographies.
  • Factor B can, under certain conditions, bind to hydrophobic interaction chromatography resins, and be desorbed therefrom using buffer systems with a low ionic strength.
  • Such chromatography uses a matrix, most commonly of agarose or polymeric type, onto which are grafted ligands such as phenyl, octyl, butyl, methyl or else hexylamine, phenylpropylamine or 4-mercaptoethyl-pyridine.
  • grafted ligands such as phenyl, octyl, butyl, methyl or else hexylamine, phenylpropylamine or 4-mercaptoethyl-pyridine.
  • the method of the invention can also comprise a hydroxyapatite-like chromatography.
  • the factor B can be purified using composites of calcium phosphate, fluoroapatite or hydroxyapatite as solid phase.
  • the method of the invention may further comprise an immunoaffinity chromatography, which uses, for example, the antibodies or aptamers immobilized on a chromatography matrix.
  • the matrices are generally resins that have been pre-activated, for example with epoxy (Sepharose 6B1, EAH Sepharose 4B, Amino Sepharose 6 FF, 6-AKS Sepharose 4FF, Toyopearl AF Epoxy, Toyopearl AF amino, Toyopearl AF Tresyl).
  • the unbound impurities are washed away using suitable buffer systems (citrate buffer, phosphate buffer or HEPES, for example), and the factor B is then eluted using, for example, high concentrations of chaotropic salts (of lithium bromide, thiocyanate type), of low-pH (glycine, pH 2-3) or high-pH (TRIS, pH 9) buffer systems.
  • suitable buffer systems such asrate buffer, phosphate buffer or HEPES, for example
  • chaotropic salts of lithium bromide, thiocyanate type
  • the method of the invention may also include other treatments, such as a delipidation.
  • Delipidation consists in removing, preferably upstream or as early as possible in the method, lipid impurities from protein solutions by means, for example, of precipitation or of adsorption.
  • Suitable precipitating agents include, for example, polyethylene glycols (PEGs) or ammonium sulphate, while silica powders (“fumed silica”, Aerosil 200, Aerosil 380), dextran sulphates or fluorocarbons (Freon-113) are suitable adsorbents.
  • proteolytic impurities namely the enzymes, for example the proteases or glycosidases
  • This removal or inhibition is preferably carried out as early as possible, more preferably upstream of the purification method.
  • a chromatographic method can be used in which protease inhibitors are immobilized on matrices of agarose or polymeric matrix type.
  • a filter which retains some of the proteolytic impurities may also be used.
  • protease inhibitors may, for example, be benzamidine, 4-aminobenzamidine, benzamidine hydrochloride and its derivatives, lysine and its derivatives, 6-aminohexanoic acid ( ⁇ -aminocaproic acid) and its derivatives, trans-4-aminomethylcyclohexanecarboxylic acid (tranexamic acid) and its derivatives, 4-(2-aminoethyl)benzenesulphonyl fluoride (AEBSF) and its derivatives, (4-aminophenyl)methanesulphonyl fluoride (APMSF) and its derivatives, 3,4-dichloroisocoumarin (DCI) and its derivatives, acetyl-leucyl-leucyl-arginal (leupeptin) and its derivatives, aprotinin and its derivatives, soybean trypsin inhibitor and its derivatives, ⁇ 2-antiplasmin and its derivatives, thrombin inhibitors
  • the proteolytic impurities can be inhibited by adding one or more protease inhibitors of the group mentioned above, in particular antithrombin III and C1-inhibitor, and then by removing the enzyme/inhibitor complex by means of subsequent purification steps.
  • immobilized dyes such as Cibacron Blue F3GA or its derivatives, other triazine dyes, such as Procion Red HE-3B and its derivatives, or Procion Green H-4G and its derivatives, Reactive Red 120 and its derivatives, Reactive Green 19 and its derivatives, Reactive Yellow 86 and its derivatives, or Reactive Orange 14 and its derivatives, immobilized on matrices of agarose or polymeric matrix type.
  • the factor B preparation that is of use in the invention has generally undergone at least one step of removing or inactivating at least one infectious agent.
  • infectious agents mention may be made of viruses and NCTAs (non-conventional transmissible agents) such as the prion.
  • Viral inactivation often comprises treatment with chemical products, for example with solvent, detergent, and/or with heat, for example by pasteurization.
  • Nanofiltration is also of use for removing an infectious agent.
  • the method comprises at least one treatment with solvent and detergent, and a nanofiltration.
  • Pasteurization refers to methods which expose the liquid factor B compositions to a temperature of 60° C. for at least 10 h.
  • the treatment with solvent and/or detergent comprises in particular treatment with tri-n-butyl phosphate TnBP and/or a detergent which is chosen from Triton X-100, Tween (preferably Tween 80) and sodium cholate.
  • TnBP tri-n-butyl phosphate
  • a detergent which is chosen from Triton X-100, Tween (preferably Tween 80) and sodium cholate.
  • the treatment is generally carried out at 25° C. for at least 6 h.
  • Nanofiltration generally refers to the filtration of a factor B solution through a filter with a pore size of less than 80 nm.
  • Available filters are, for example, cuprammonium regenerated cellulose filters PLANOVATM 75 nm, PLANOVATM 35 nm, PLANOVATM 20 nm or PLANOVATM 15 nm, BioEX (Asahi Corporation), Ultipor DV 50 or DV 20 (Pall Corporation), Virosart CPV (Sartorius), Viresolve NFR or NFP (Millipore).
  • a nanofiltration is carried out after the anion exchange chromatography step (iv).
  • the purified factor B fraction is filtered on a sequence of filters with a pore size of 20 nm and 15 nm.
  • the factor B preparation obtained is formulated with suitable excipients and stabilizers. These may be diluents, cyroprotective agents, lyoprotective agents, etc.
  • sugars sucrose, trehalose, glucose, lactose, etc.
  • polyols mannitol, sorbitol
  • amino acids glycine, arginine, histidine, alanine
  • Surfactants of polysorbate Teween 20 or Tween 80 for example
  • poloxamer for example poloxamer 188) or polyethylene glycol type can also be added.
  • Antioxidants for example methionine, monothioglycerol, glutathione, citric acid, ascorbic acid, sodium metabisulphite and sodium sulphite may also be added.
  • Buffer substances may also be used, for example in the form of carbonate, phosphate, citrate, acetate, borate, trimethamine [(2-amino-2-(hydroxymethyl)propane-1,3-diol), TRIS], glycine and lysine.
  • the liquid factor B formulation can undergo desiccation if necessary, in order to obtain a solid form.
  • Desiccation is a process for extensive removal of water. It is a dehydration aimed at removing as much water as possible. This phenomenon may be natural or forced. This desiccation can be carried out by means of lyophilization, spray-drying or cryo-spray-drying techniques.
  • the preferred method for obtaining the solid form of the composition for pharmaceutical use according to the invention is lyophilization.
  • Lyophilization methods are well known to those skilled in the art, see, for example Wang et al., Lyophilization and development of solid protein pharmaceuticals, International Journal of Pharmaceutics, Vol. 203, p. 1-60, 2000.
  • the degree of moisture is less than or equal to 3% by weight, preferably less than or equal to 2.5%, more preferably less than or equal to 2%, preferably less than or equal to 1.5%.
  • the solid composition can be dissolved in water for injection (WFI) or in a reconstituting solvent, so as to obtain a formulation for therapeutic use.
  • WFI water for injection
  • a reconstituting solvent so as to obtain a formulation for therapeutic use.
  • a factor B concentrate is prepared according to the steps represented in the attached figure.
  • Cryosupernatant is prepared by thawing fresh plasma frozen at a temperature between 1° C. and 6° C. (cryoprecipitation). After centrifugation, the cryoprecipitate is recovered in order to produce the fibrinogen, von Willebrand factor and factor VIII concentrates. The supernatant is collected, of which a part is subjected to a purification step on DEAE-Sephadex in order to bind the vitamin K-dependent factors, such as protein C, factor VII and factor IX. The fraction not retained on DEAE-Sephadex is mixed with the remaining cryosupernatant so as to constitute solution A.
  • the factor B concentration in the fraction is approximately 100-150 ⁇ g/ml (for a recorded plasma concentration of 200 ⁇ g/ml).
  • Solution A is adjusted to a pH of between 5.5 and 6.5, and preferably to 6.0.
  • the adjusted fraction is then subjected to a chromatography on a Heparin Sepharose FF column or other chromatographic support onto which a heparin ligand is grafted. Most of the plasma proteins are found, not retarded, in the chromatography filtrate.
  • the factor B is eluted by increasing the ionic strength of the column equilibration buffer. The eluted fraction is diluted, and then chromatographed on a strong cation exchange column of SP Sepharose FF type or equivalent.
  • the proteins weakly adsorbed onto the gel are eluted by increasing the ionic strength of the column equilibration buffer.
  • the factor B is eluted by even further increasing the ionic strength of the column equilibration buffer.
  • the eluted fraction is then subjected to a viral inactivation step by solvent/detergent treatment (Polysorbate 80 and TnBP) which is effective on enveloped viruses.
  • the fraction is then diluted, adjusted to a pH of 6.0 (assay 1) or 6.5 (assay 2), and then subjected to a chromatography on a strong anion exchange column of Q Sepharose FF type or equivalent, equilibrated at pH 6.0 or 6.5.
  • the factor B is eluted by increasing the ionic strength of the column equilibration buffer.
  • the factor B fraction obtained at this stage is of very high purity and can be subjected to a step of viral removal by nanofiltration, and then concentrated and formulated by ultrafiltration.
  • the factor B is assayed conventionally by means of an immunoenzymatic method (ELISA) with commercial reagents (Diagnostica Stago). Briefly, the factor B to be assayed is captured by an anti-human factor B antibody immobilized on a solid phase. The factor B bound is then recognized by a peroxidase immunoconjugate. The amount of peroxidase bound is measured by its activity on the substrate ortho-phenylenediamine in the presence of aqueous hydrogen peroxide. The strength of the coloration, after the reaction has been stopped with a strong acid, depends on the amount of factor B initially present in the sample. The factor B thus assayed is called “factor B antigen” or “Ag”.

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US13/510,140 2009-11-16 2010-11-15 Method for purifying factor B Expired - Fee Related US8729244B2 (en)

Applications Claiming Priority (3)

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FR0958047A FR2952639B1 (fr) 2009-11-16 2009-11-16 Procede de purification de facteur b
FR0958047 2009-11-16
PCT/FR2010/052426 WO2011058286A1 (fr) 2009-11-16 2010-11-15 Procede de purification de facteur b

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EP (1) EP2501401A1 (fr)
AR (1) AR079033A1 (fr)
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WO2014035876A1 (fr) * 2012-08-27 2014-03-06 William Marsh Rice University Compositions de facteurs b du complément désactivé à la chaleur et procédé
FR3004451B1 (fr) * 2013-04-11 2015-12-11 Lab Francais Du Fractionnement Procede de preparation d'une solution de proteine c viralement securisee par une double etape de nanofiltration
CN108473530B (zh) * 2015-10-15 2022-12-23 普莱泽玛科技有限公司 从血浆中提取蛋白质的方法

Citations (4)

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Publication number Priority date Publication date Assignee Title
FR2234312A1 (fr) 1973-06-19 1975-01-17 Kabi Ab
US4675384A (en) * 1983-04-01 1987-06-23 Rhone-Poulenc Recherches Fractionation/purification of plasma by ion exchange chromatography
WO2008106644A2 (fr) 2007-03-01 2008-09-04 Advanced Vision Therapies, Inc. Traitement de maladies caractérisées par une inflammation
WO2008113589A1 (fr) * 2007-03-20 2008-09-25 Csl Behring Gmbh Procédés pour la production à échelle industrielle de préparations thérapeutiques de facteur h du complément à partir du plasma humain

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2234312A1 (fr) 1973-06-19 1975-01-17 Kabi Ab
US4675384A (en) * 1983-04-01 1987-06-23 Rhone-Poulenc Recherches Fractionation/purification of plasma by ion exchange chromatography
WO2008106644A2 (fr) 2007-03-01 2008-09-04 Advanced Vision Therapies, Inc. Traitement de maladies caractérisées par une inflammation
WO2008113589A1 (fr) * 2007-03-20 2008-09-25 Csl Behring Gmbh Procédés pour la production à échelle industrielle de préparations thérapeutiques de facteur h du complément à partir du plasma humain

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Title
Cheung, AK, and Parker CJ (1992) Journal of the American Society of Nephrology 2, 1328-1337. "Modualtion of Complement Activation on Hemodialysis Membranes by Immobilized Heparin". *
GE HEalthcare Data File 18-1172-88 AB Q Sepharose High Performance SP Sepharose High Performance 2006. *
International Search Report, PCT/FR2010/052426, mailed Mar. 9, 2011 (3 pages).
Lambris et al. "Release of endogenous C3b inactivator from lymphocytes in response to triggering membrane receptors for b1H globulin" 1980 J. Exp. Med. 152 1625-1644. *
Lambris, JD and Muller-Eberhard HJ (1984) Journal of Biological Chemistry 256, 12685-12690. "Isolation and Characterization of a 33,000-Dalton Fragment of Complement Factor B with Catalytic and C3b Binding Activity". *
McKay et al. "The interaction of heparin with plasma proteins" 1980 J. Lab. Clin. Med. 95, 69-80. *
Nakamura et al., "Purification and Properties of Intracellular Clotting Factor, Factor B, from Horseshoe Crab (Tachypleus tridentatus) Hemocytes," J. Biochem., 99: 847-857 (1986).
Pang, A.S.D. and W.P. Aston, "The Alternative Complement Pathway in Bovine Serum: The Isolation of a Serum Protein with Factor B Activity," Immunochemistry, 15:529-534 (1978).
Williams, S.C. and R.B. Sim,"Dye Ligand Affinity Purification of Human Complement Factor B and beta2 glycoprotein I," Journal of Immunological Methods, 157:25-30 (1993).
Williams, S.C. and R.B. Sim,"Dye Ligand Affinity Purification of Human Complement Factor B and β2 glycoprotein I," Journal of Immunological Methods, 157:25-30 (1993).

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FR2952639A1 (fr) 2011-05-20
US20120232253A1 (en) 2012-09-13
AR079033A1 (es) 2011-12-21
WO2011058286A1 (fr) 2011-05-19
FR2952639B1 (fr) 2013-08-30
EP2501401A1 (fr) 2012-09-26

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